The invention relates to liquid chromatography deposition media compatible with infrared (IR) analysis.
In the field of chemical analysis concerned with identification of organic compounds in complex mixtures, liquid chromatography is a widely used separation process which relies on the differential adsorption properties of organic molecules. Typically an organic mixture in a specific solvent is added to the top of a tubular column which has been packed with a fixed bed of adsorbent material providing surface area onto which substances may be adsorbed. As the solvent and solute mixture descend through the column, more strongly adsorbed compounds coat the packed bed surfaces, referred to as the stationary phase. The less strongly adsorbed substances, proceed through the column, along with the solvent. Ideally, the substances are progressively retarded into well separated segments. The eluted separated components of the mixture are discharged from the other end of the column along with solvent or eluent. Properly separated, the organic compounds come out of the column at intervals spaced by relatively pure solvent effluent.
For high performance liquid chromatography (HPLC), narrow columns known as microbore columns, may be employed to reduce solvent consumption and promote high solute concentrations. A commercially available microbore HPLC column 50 cm long with a 1 mm internal diameter is loaded with 10 micrometer (.mu.m) silica beads. In normal phase chromatography, hydrocarbon solvents such as hexane and dichlormethane are used in the mobile phase. In reversed phase chromatography, polar organic solvents such as methanol are used in combination with water.
Once separated by chromatography, the individualized organic substances can be analyzed for identification by a variety of techniques, including, for example, IR spectroscopy, mass spectrometry, nuclear magnetic resonance, differential refractometry, heat of absorption detection and modified hydrogen flame ionization detection. In particular, the high scan speed and sensitivity of Fourier transform infrared (FTIR) spectroscopy has greatly facilitated the recording of characteristic infrared spectra of the individual components of mixtures separated by chromatographic techniques. Organic molecules in general contain interatomic bonds which exhibit characteristic vibrational frequencies, many of which happen to be in the mid IR region. These can be identified in the absorption spectrum of the material.
Interfacing HPLC with FTIR is hampered by infrared absorption of the extraneous solvent remaining in the mobile phase after separation. Two types of interfaces have appeared in the literature: (1) flow cells, which allow recording the IR spectra while the HPLC effluent flows by a window transparent in the infrared and (2) solvent deposition systems which involve transfer and elimination of the solvent on a medium compatible with infrared sampling.
In flow cells, the spectral contribution including spectral masking produced by the solvent material, which is still present at full strength, must be taken into account. Thus, analysis by the flow cell method is limited to solvents which happen to be transparent in wide regions of the infrared spectrum and even then, some areas of the spectrum will remain opaque resulting in loss of information and sensitivity.
Solvent deposition designs, on the other hand, involve depositing concentrated portions of HPLC effluent onto a collection medium and thoroughly evaporating the solvent. The deposits are then exposed to IR and the spectrum is measured and analyzed. Deposition surfaces thus offer the advantage of not only eliminating the solvent, but also leaving the solute on the collection surface as a record of the chromatographic separation. Collection media for analysis of deposits by transmission or reflection are limited to materials which do not have significant infrared absorbances of their own, the most practical such material being potassium bromide (KBr), which happens to be transparent over the entire intermediate infrared range examined in IR spectroscopy.
In general, the design of an interface for use with organic solvents common to normal phase HPLC has been possible since the solvents may be judiciously chosen to provide regions of IR transparency when used with flow cells, or can be evaporated during or after deposition on conventional infrared sampling media such as KBr. The use of aqueous solvents in the commonly employed reversed phase mode of HPLC, however, further complicates either design because water absorbs strongly over broad regions of the infrared, is of comparatively low volatility, thus frustrating evaporation, and readily dissolves KBr.